IT8224948A1 - Modified vaccine virus and methods for its production and use - Google Patents

Description

DESCRIPTION

This invention deals with modified vaccine virus, the methods for the production and use of the same and other modified and unmodified microorganisms, as well as? of certain DNA sequences, produced or involved in quality? of intermediaries in the production of modified vaccine virus. particularly, the invention deals with the vaccine virus in which the genome naturally present in the virus? been altered (the so-called vaccine mutants) and of the methods used in the production and use of such vaccine mutants, as well as? of other unmodified and genetically modified microorganisms, and of certain DNA sequences, produced or involved in quality? of intermediaries in the production of vaccine mutants.

The vaccine virus? the virus prototype of the poxvirus family which, like other members of the poxvirus group, is distinguished by its large size and complexity. The DNA of the vaccine virus? equally large and complex. For example, vaccine DNA? measuring approximately 120 megadaltons, compared to just 3.6 megadaltons for the SV40 monkey virus. The vaccine DNA molecule? double chain with helical winding of the ends ?, so? DNA denaturation leads to the formation of a single-stranded ring. The physical map of vaccine DNA? was built with the help of a munero of different enzymes called restriction and various maps of the kind are reported in the monograph by Panicali and coll, published in J.?Virol.?37:1000-1010, 1981, which cites the existence of two main DNA variants in the WR strain of the vaccine virus (ATCC No. VR 119), namely the pi? commonly used for the study and characterization of poxviruses. The difference between the two variants is that the. variant S ("small, ie short) (ATCC No. VR 2034 dep.'18-11-81) has a deletion of 6.3 megadaltons that is missing in the DNA of variant L (" large ", i.e. long) (ATCC No. VR 2035 dep. 18-11-81) .- The aforementioned publication presents the maps resulting from the treatment of these variants with restriction enzymes such as Hind III, Ava I, Xho I, Sst I and Sma I.

The vaccine virus belongs to the eukaryotic group reproduced entirely within the cytoplasm of the host cell. It is a lytic virus, that is, a virus whose intracellular replication leads to cell lysis. deemed non-oncogenic. It has been used for about 200 years in vaccines used for inoculation against smallpox and in medical circles the properties are well known. of the virus used in the vaccine. 'Although inoculation with the vaccine virus involves a certain risk, these risks are generally well known and defined and the virus itself? considered to be relatively benign in nature.

The invention essentially concerns the modification of the genome naturally present in the vaccine virus in order to produce vaccine mutants through the rearrangement of the natural genome, the elimination of DNA from the genome, and / or the insertion into the natural vaccine genome of the DNA capable of modifying the natural genome ("foreign DNA"). Foreign DNA can be naturally present in the vaccine virus or can? be of synthetic or even natural origin, but in an organism other than the vaccine one. To the extent that foreign DNA? carrier of genetic information, exists-, the possibility? to introduce the information itself into a eukaryotic organism by means of the modified vaccine virus. In other words, the modified virus represents a relatively harmless vector of eukaryotic cloning following the suppression, insertion or rearrangement of the genetic information. ' Since? the virus replicates within the cytoplasm of the infected cell, the modified vaccine virus constitutes a singular eukaryotic cloning vector without equal among those studied in the past approximately, equal to about 10 kbp (coupled kilobases). Since? the central segments of DNA are similar in all poxviruses, while the terminal ones differ more, it seems likely that the central segment is responsible for the functions common to all viruses, for example replication, while the terminal segments would be responsible for other characteristics , for example the pathogenicity, the spectrum of infectivity? and so? Street. Having to modify, a genome for the rearrangement or elimination of DNA fragments or for the introduction of exogenous DNA fragments with the purpose of arriving at a stable and viable mutant,? obvious that the fraction of the natural -DNA that one wants to rearrange, suppress or break up through the introduction of exogenous DNA must be non-essential to vitality? and stability host organism, in this case the vaccine virus. In the WR strain of the vaccine virus there are non-essential segments of the genome, for example in the trait preserved in variant L, but suppressed in variant S, or in the Hind III F fragment of the genome.

The modification of the vaccine virus through the incorporation of exogenous genetic information finds an example in the modification of the WR strain of the virus itself in the Hind III F fragment, consisting of the incorporate? cos? the antigenic determinant in vivo.

A further advantage of vaccine mutants used as vectors in eukaryotic cells, as vaccines or for the production of biological compounds other than antigens, lies in the possibility? to modify after the translocation process the proteins resulting from the transcription of the exogenous genes introduced into the cell by the virus. Such post-translocative modifications, for example the glucosylation of proteins, are unlikely within a prokaryotic system, being instead possible in eukaryotic cells which have the additional enzymes necessary for such modifications. A further advantage of vaccine mutants for inoculation use lies in the possibility? to broaden the antibody response by incorporating replicated pairs of the antigen gene or additional genetic elements into the mutant to stimulate the immunological reaction, or by using potent activators in the modified virus. A similar advantage is found in the production of biological substances other than antigen.

Resuming now an exam pi? detail of the vaccine genome, the double helical DNA chains are characterized by inverted terminal repetitive elements, each of the length of 8.6 megadaltons at? marne the antigenic potency. On the other hand, vaccines derived from attenuated living organisms always carry the risk that the attenuated organism can resume pathogenic virulence. antigenic of the pathogenetic organism, you avoid the possibility? of a reversion to the pathogenetic state, since the vaccine virus contains only the gene encoded for the antigenic determinant of the morbid organism and not those genetic fractions of the organism which are responsible for the replication of the pathogen.

The present invention also offers advantages over modern technology based on genetic engineering intended to produce antigen by means of a recombinant prokaryotic organism containing plasmids as the expression of a foreign antigenic protein. , such a technology requires the production of numerous PR cells. karyotic recombination followed by purification of the resulting antigenic proteins. On the contrary, the modified vaccine virus for inoculation use according to the subject invention replicates in the individual inoculated for the purpose of immunization, increasing at the present time.

Such a discovery involves a variety? of useful consequences, including (A) innovative methods, for the vaccination of mammals susceptible to the vaccine virus to induce in them an antibody response to the antigens encoded for the foreign DNA introduced into the vaccine virus, (B) innovative methods for the production through eukaryotic cells of biological products other than antigens, and (C) innovative methods for the introduction into individuals or populations of humans or animals, of missing genes or genetic material capable of modifying, replacing or repairing defective genes in such individuals or populations ? tions .-- The vaccine mutants suitably modified as carriers of exogenous genes expressed in the host organism in the form of determining antigenic factors capable of stimulating the production of anti? bodies towards antigen, constitute a new type of vaccines free from the drawbacks of common vaccines derived from killed or living but attenuated organisms. What? for example, the production of vaccines from killed organisms requires the growth of large quantities of the organism followed by a treatment aimed at electively destroying its capacity? infectious, not least within that fragment of a herpes simplex virus type I (HSV) gene responsible for producing

thymidine kinase (TK). The TK? an enzyme suitable for a

phosphorylate thymidine, a nucleoside substance, to form the related mono? phosphorylated nucleotide which is then incorporated into DNA.

The HSV TK gene constitutes DNA foreign to the vaccine virus, which according to the current invention? appropriate to introduce into the vaccine virus for a number of reasons. First, the availability? of the gene? relatively high in the herpes simplex virus DNA fragment resulting from cleavage with the Barn HI endonuclease, as published by

Colbere-Garapin et al, in Proc. Nati. Acad. Ski.

USA 76: 3755-3759, 1979. Second,? already? Note

The introduction of this fragment of the HSV Barn HI in plasmids and eukaryotic systems, for example from the publications of Colbere-Garapin et al., Loc. cit. as well? by Wigler and coll. Cell 1J_: 223-232, 1977. Thirdly, experience shows that if the HSV TK can? be introduced in the form of an exogenous gene into a eukaryotic system - an expression that requires precise and very faithful translocation and transcription of the genetic locus then other exogenous genes can be similarly introduced and expressed.

For a better understanding of the invention, reference is made to the attached graphs in which,

Figure 1? a map of the aforementioned L and S variants of the vaccine WR strain, determined with the aid of Hind III as a restriction enzyme, which illustrates the deletion of the sequences in the C terminal fragment of the L variant, the deletion being outside the terminal zone of repetition of the genome. The DNA sequences cos? deletion are an exclusive feature of the L structure and since? the growth of the S and L variants (dep. 18-11-81)? identical, the deletion zone must be non-essential;

Figure 2 illustrates more? in detail the vaccine Hind III F fragment, including two other restriction sites, i.e. Sst I and Barn HI, of which at least the latter offers a locus suitable for introducing exogenous DNA into the vaccine Hind III F fragment, without any interference with the essential vaccine genes; Figures 3 A - C schematically illustrate a method for introducing the HSV TK gene into the vaccine Hind III F fragment;

Figure 4 constitutes a restriction map of certain vaccine mutants produced according to the present invention, where the position of the HSV TK inserted in the Hind III F fragment in two mutants of the Virus is indicated in detail, marked by the initials VP-1 and VP- 2 (dep. 18-11-81);

Figure 5 summarizes in the form of a mirror certain techniques useful in sorting viruses potentially suitable for recombination, to determine the presence or absence of the HSV TK gene; And

Figures 6 A - C are restriction maps of the left terminal segment of the WR vaccine genome illustrating the correlation within the various restriction fragments and the characteristic DNA sequence found in variant L and suppressed in variant S.

Referring now to figure 1, if we want to subject the L and S variants of the vaccine virus to the action of Hind III, a well-known restriction enzyme already? available on the market, the viral genomes will be split respectively into 15 or 14 segments marked by the letters A to 0, being A the most? -long and 0 the more? short. The aforementioned publication by Panicali and coll., J. Virol. 37i1000-1010, 1981, describes and illustrates the electrophoretic separation of the restriction fragments. The F fragment obtained in this way from one of the L or S variants has a molecular weight of 8.6 megadalton.

The location of the fragment F? marked in the restriction map presented in Figure 1 attached to the patent application, while another restriction map of the fragment F? shown in Figure 2. The Hind III restriction enzyme recognizes the nucleotide sequence -AAGCTT- and cleaves the DNA between the two adjacent adenosine groups, resulting in * 'sticky stump' fragments and the AGCT- sequence. Since? the quantities of the vaccine Hind III F fragment necessary for the manipulations envisaged by the present invention exceed those conveniently obtainable by restricting the vaccine genome, the F fragment must be inserted into a plasmid vector for cloning for growth.

Pi? precisely, the Hind III F vaccine fragment produced in this way is conveniently introduced into the pBR 322 plasmid cleaved only once by a number of restriction enzymes, including the Hind III one. The plasmid pBR 322 was first described by Bolivar et al, in Gene ??: 95-113, 1977, ed? currently available in the US market from a variety of suppliers.

The location of the Hind III cleavage site on the pBR 322 plasmid? marked in Figure 3A in relation to the cleavage site of two other restriction enzymes, i.e. the Eco RI and the Barn HI. By cleaving the pBR 322 plasmid with Hind III and mixing the resulting cleaved DNA with the Hind III vaccine fragment F to then bind the fragments with the DNA ligase T, as indicated in Figure 3A, the fragment F is incorporated into the plasmid, resulting in a new plasmid pDF 3, schematically illustrated in Figure 3B, having a molecular weight equal to about 11.3 magadalton. The Hind III F vaccine fragment comprises approximately 13 paired kilobases, versus 4.5 kbps found in the pBR 322 segment of the pDP 3. The DNA ligase? an enzyme available on the market and its conditions of use in the modality? indicated are well known to those skilled in the art.

The pDP 3 plasmid is now introduced into a microorganism such as Escherichia coli (E. coli) by means of transformation with the aim of replicating the Hind III F fragment and recovering a greater quantity of the fragment itself. These techniques for the cleavage of a plasmid in order to produce linear DNA with binding terminals, to then insert the exogenous DNA with complementary terminals in order to form a replicon (in this case the pBR 322 containing the Hind III F vaccine fragment) are they already? known and the same applies to the insertion of the replicon into a microorganism by means of transformation

(see U.S. Patent 4,237,224).

The unmodified pBR 322 plasmid confers resistance to ampicylin (Amp-) as well as? to tetracycline (TetR) to its host organism, in this case E. coli. However, since? the Hind III breaks the plasmid pBR 322 in the TetR gene, the introduction of the Hind III F vaccine fragment destroys the R gene

Tet, getting lost in this way? resistance to tetracycline. Therefore, E. coli transformants containing the pDP 3 plasmid are distinguished from untransformed E. coli by the simultaneous presence of ampicillin resistance and susceptibility. to tetracycline. It is these E. coli bacteria transformed with pDP 3 that grow greatly to obtain large amounts of the pDP. 3.

The conditions of plasmid growth in E. coli bacteria are well known to those skilled in the art, for example from the Clewel report published in the journal J. Bacteriol. 110: 667-676, 1972. Equally well known are the techniques for isolating the plasmid grown from the bacterial host E. coli, as described for example by Clewel et al, in Proc. Nati. Acad. Sci. USA 62: 1159-1166, 1969.

By analogy, the plasmid pBR 322 can be suitably cleave by treatment with the Ban HI restriction enzyme, then deriving a modified plasmid by inserting a Ban HSV TK fragment, as specified in the cited article by Colbere-Garapin et al., loc. cit. The modified plasmid containing the Bam HI fragment complete with HSV TK gene can? be re-introduced in E. coli with techniques already? known and transformed bacteria can be cultured to increase large quantities of the plasmid. Thereafter, the enhanced recombinant Barn HSV plasmid TK-pBR 322 can be performed. cleave with Barn HI in order to isolate the Barn HI fragment containing the HSV TK gene with the same techniques already? notes and previously cited in relation to the growth of the Hind III F. vaccine fragment.

In order to construct a recombinant plasmid having the Barn HI HSV TK fragment incorporated into the Hind III F vaccine fragment, the pDP 3 plasmid is then exposed to partial restriction with the Barn Hi, so that only one of the two Barn HI cleavage sites within the plasmid is cleaved, i.e. either the Barn HI site within the Hind III F fragment or the Barn HI site within the pBR 322 fraction of the pDP 3 plasmid, as illustrated in Figure 3B. The cleaved DNA, now linear, is then combined with the purified Barn HSV TK fragment. The linear segments are combined and linked by treating them with the T DNA ligase according to already techniques. Note.

The combination of the HSV TK Barn fragment with the cleaved pDP 3 plasmid constitutes a random event or a statistical occurrence potentially capable of producing numerous species formed by different combinations of the fragments present in the mixture, each of them equipped with identical 'sticky stumps'. Therefore, one of the possibilities? consists of a simple rejoining of the ends? cleaved of the plasmid pDP 3 to recreate the plasmid ring.-Another possibility? consists of the junction of two or more? fragments

Barn HSV TK in one of two orientations. Furthermore, the HSV TK Barn fragment (or a multiple) can be combined with the linear DNA of a pDP 3 plasmid cleaved at the Barn HI site within the pBR 322 tract, also in one of the two orientations, or one or more? Barn HSV TK fragments can be combined in either orientation with the linear DNA of the pDP 3 plasmid cleaved at the Barn HI site within that tract of the pDP 3 plasmid that constitutes the Hind III F vaccine fragment.

In order to allow the identification and separation of these various possibilities, the binding products are inserted into a single-celled microorganism such as E. coli with techniques that are already in use. notes and described above. E. coli bacteria treated in this manner are then cultured on a medium containing ampicillin. If the bacteria contain the plasmid, they will be resistant to ampicillin as all such plasmids contain the pBR 322 gene which confers resistance to ampicillin. ' Therefore, the surviving bacteria are all transformation products, suitable for a further sorting to determine the possible presence or absence of the Barn HSV TK fragment.

For this purpose, bacteria endowed with a TK gene must be identified by hybridization with radiolabeled TK DNA. Where the TK gene? present in bacteria, the radiolabeled TK DNA former? a hybrid with the plasmid fraction present in the bacterium. Being the radioactive hybrid, the colonies containing the plasmidic TK can be tested with the aid of autoradiography. In turn, TK-containing bacteria can be cultured. Finally, the bacteria containing the plasmids endowed with the TK incorporated in the pBR 322 tract can be identified and separated from those carrying the TK fragment within the Hind vaccine fragment.

III F by restriction endonuclease analysis. Pi? particularly, bacteria that survive cultivation on ampicillin inoculated nutrient agar plates are transferred in part to a nitrocellulose filter by contact between the filter and plate. Those bacteria that remain on the plate are recultivated while those transferred to the nitrocellulose filter with the aim of creating a replica of the original plate are then subjected to a treatment to denature the DNA. sodium hydroxide, which is followed by neutralization and washing, after which the denatured DNA present on the nitrocellulose filter is hybridized by treatment with radioactive 32p-labeled HSV Barn TK. After this treatment, the nitrocellulose filter is exposed on a roentgenographic film which becomes opaque in the areas corresponding to the hybridization with the radiolabelled HSV TK Barn. The roentgenographic impression on the opacified film is then compared with the original plate and the colonies grown on that original plate in correspondence with the colonies that gave rise to the opacities. on the film, colonies containing a plasmid having the Barn HSV TK are identified.

Finally, to be able to distinguish the bacteria containing the plasmid whose gene Barn HSV TK? been incorporated into the pBR 322 section from those in which the HSV TK Barn? present in the plasmid fragment F, small bacterial cultures are set up to isolate the plasmids by means of minilitic techniques already? notes and described in the report by Holmes et al.,

Anal.?Bioch. 114: 193-197, 1981. The plasmids are then fermented with the Hind III restriction enzyme which breaks the plasmid ring at the two points where the F fragment was originally joined with the pBR 322 DNA chain. The molecular weight of the fermentation product is then determined by electrophoresis on agarose gels, the migration distance on the gel being an index of the molecular weight.

If the fermented plasmid contains the Barn HSV TK fragment or a multiple in the F segment, the gel will highlight? a pBR 322 fragment and another fragment having a molecular weight higher than that of the F fragment by the molecular weight of the segment 0 of the Barn HSV TK DNA segments present therein. Conversely, when the HSV TK Barn? present in the pBR 322, the electrophoresis evidenzier? a fragment F of the normal molecular weight in pi? to another fragment exceeding pBR 322 for the molecular weight of the Barn HSV TK fragment or fragments included therein. The bacteria modified with Barn HSV TK in the plasmid fraction pBR 322 are discarded, leaving instead those modified in the fragment F of the plasmid. The latter are the plasmids used for the vaccine incorporation of the fragment? Barn HSV TK.

How already? said, the combination of the DNA fragments intended to regenerate a plasmid constitutes a random event that can? result in a variety? of plasmid structures bearing the HSV TK Barn in the F.

With the purpose of determining the orientation of the Barn HSV TK fragment within the F fragment, as well as? the number of such HSV TK Barn fragments possibly present, the plasmids are recovered from all those bacterial colonies which are endowed with the Barn HSV TK fragment in the F fragment of the plasmid.

The aforementioned mini-lysis technique is employed in this regard. The plasmids are then subjected to restriction analysis again, this time with the aid of the commercially sourced restriction enzyme Sst I. Since? each fragment Barn HSV TK bears a restriction site Sst I and since? the vaccine fragment F likewise bears a single restriction site Sst I (see the respective representations of such fragments in Figures 3A and 3B), ci? given to identify a different number of fragments of different molecular weight by means of agar gel electrophoresis? physical, since the number and molecular weight of the segments are a function of the orientation of the Barn HSV TK fragment within the landslide F and of the number of Barn TK fragments present. Is the orientation of the Barn TK fragment within the F fragment? identify thanks to the asymmetrical arrangement of the 3am HSV TK fragment in relation to the relative Sst I site (see figure 33).

For example, in the course of the experiments discussed here it was possible to identify six bacterial colonies, each bearing one or more? Barn HSV TK fragments within the F fragment of the plasmid, among those capable of transforming E. coli. Following a restriction analysis of the bacterial plasmids as outlined above, two of the recombinant plasmids were selected for further examination due to the fact that the Barn HSV TK fragments within the F fragment were oriented in the opposite direction.

At this point it should be pointed out that the introduction of the HSV TK gene into the vaccine fragment F, as discussed in greater detail above, simply serves as an example as one of several possible techniques for modifying the vaccine genome with the aim of reaching mutants desired vaccines. Therefore, the introduction of the same exogenous gene into some other section of the vaccine genome, or the introduction of different genetic material into the vaccine fragment F or some other fragment, may be necessary. the modification of the scheme already? discussed by way of example, for the identification of recombinant organisms.

For example, when the vaccine variant L is digested with Ava I, the result is an H fragment located entirely in the deletion region of variant S (see Figure 6 A and related discussion below). Fragment H contains Bam HI sites that allow the introduction of the HSV TIC gene. These recombinants of the H fragment are suitable for identification with the same techniques suggested for the identification of the recombinants of the F-HSV TK fragment.

In fact, there are schemes for the construction and identification of the recombinants of the F-HSV TK fragment which represent alternatives to the already project. discussed in detail for illustration. For example, the HI Barn site in the pBR 322 you can? suppress by cleaving the plasmid with Barn HI and proceeding to the treatment with DNA polymerase I as 'filler' of the sticky stumps. This product is then cut with Hind III and the linear fragment is treated with alkaline phosphatase to prevent recirculation of the plasmid following ligation. However, the foreign DNA and in particular the vaccine fragment F of the Hind III can be carried out. bind to the treated pBR 322 and the resulting plasmid will be? recirculated. The treatment with Barn HI simply serves to cleave the plasmid within the relative stretch of the vaccine fragment F. Subsequent treatment of the cleavage product with alkaline phosphatase and ligation with the Barn Hi HSV TK fragment will produce? recombinants with a? high efficiency, so? the recombinants will be able to undergo sorting by means of cleavage with restriction endonuclease and gel electrophoresis. AND? this is a technique which eliminates the costly steps of discrimination between recombinants having HSV TK in the pBR 322 or F fragment and colony hybridization.

Resuming now the discussion of the plasmids produced by way of example in the vaccine mutation through the introduction of HSV TK in the vaccine fragment F, the two recombinant plasmids chosen for further investigation are illustrated in Figure 3C, being identified as the first new plasmid pDP 132, bearing a Barn HSV TK fragment in the Hind III F vaccine tract, and the second new plasmid pDP 137, bearing two Barn HSV TK fragments, joined head to tail. The single fragment of the HSV TK Barn? was incorporated into the pDP .132 in the opposite direction to that in which the two Barn TK fragments were included in tandem in the pDP 137. In other terms, the region of the TK gene within the Barn HI fragment encoded for the 5 'terminal of the mRNA produced by the gene? located between the site of division and the pi? neighbor of the two corresponding Barn HI sites (see Figure 3B). Transcription of the HSV TK gene on the TK Barn fragment proceeds in the direction from the 5 'to the 3' terminal, clockwise in the pDP 132 as indicated in Figure 3C. (see Smiley et al., Virology 102: 83-93, 1980). Vice versa, since? the fragments of the Barn TK included in tandem in the pDP 137 have been incorporated in the reverse direction, sinch? the transcription of the HSV TK genes contained in it will proceed? in the opposite direction, that is, in a counterclockwise direction. The direction in which the HSV TK Barn fragment? included in the vaccine fragment Hind III F pu? be of some importance in case the HSV TK gene transcription is activated by an activation site within the F fragment itself. However, such sites of activation of the HSV exist within the Barn HSV TK fragment itself, so much so that the transcription of the HSV TK gene can? occur regardless of the direction in which the Barn HSV TK fragment and the HSV TK gene have been incorporated into the Hind III F vaccine fragment.

Those transformants of E. coli containing pDP 132 or pDP 137, to obtain greater quantities of plasinides for further processing. Once a sufficient quantity of the DNA plasmid has been isolated, enzymatic restriction by Hind III yields a modified Hind III F vaccine fragment endowed with the HSV TK gene. This modified Hind III F fragment is now being introduced into the vaccine virus by original methods, described below, to produce the entity. infectious.

To review the pre-existing knowledge, the vector currently pi? used to introduce exogenous DNA into eukaryotic cells? the SV 40. The DNA of the SV40? circular and amenable to treatment as a plasmid. In other words, the circular DNA is cleaved with a restriction enzyme, then combined with the exogenous DNA and bound. The modified DNA can? be introduced into eukaryotic cells, such as animal cells, by standardized techniques (see Hamer et al., Nature 281: 35-40, 1979). DNA? infectious and will replicate? in the cell nucleus producing viable mutation viruses. Conversely, the vaccine virus replicates in the cytoplasm of the eukaryotic cell. The purified DNA of this virus is not? infectious and does not per se lend itself to the production of vaccine mutants in the cell by analogy to SV40. Rather, one must resort to innovative techniques based on the mutation of 'wild' vaccine viruses with foreign DNA, both living and within the cell.

An unpublished communication from the applicants, written in collaboration with Eileen Nakano, describes the demonstration of the so-called 'recovery' of marking in the vaccine virus. According to these experiments, that trait of the L variant of the DNA that is ordinarily missing in the S variant can? be reintroduced ('recovered') into the S variant under appropriate circumstances.1 To this end, eukaryotic cells are treated with the living and infectious S variant of the vaccine virus together with non-infectious restriction fragments of the L variant of the DNA, making up the DNA ' unrelated to variant S, of a particular structure. Pi? precisely, that stretch of the L variant of the DNA that is to be recovered must be present within a DNA chain having sections co-linear with the DNA chain of the S fragment into which it is to be introduced. In other words, the foreign DNA to be introduced in the S variant must have both ends? of the DNA chain a DNA region that is the homolog of the corresponding sequences in the S variant. Such homologous sequences can be understood as "arms" joined to the region of the L variant of the DNA which has to be recovered with the S variant.

The mechanism of this recombination? complex and not yes? managed to achieve it in vitro so far.

Apparently, the recombination of the L variant of the DNA into the S variant consists of the pairing of homologous bases in the segments surrounding the deletion zone of the S variant. the exchanges from one DNA strand to another lead to an in vivo recombination of the DNA, capable of recovering the suppressed trait.

Such an in vivo recombination technique lends itself to the introduction of foreign DNA other than vaccine DNA into either S or L variants of the vaccine virus. Hence, the modified Hind III F fragment incorporating the HSV TK Barn fragment in quality? of DNA foreign to the vaccine virus pu? be introduced into the latter by treating the eukaryotic cells with the modified F fragment together with the infectious L and / or S variants of the vaccine virus. In this case, the portions of the F fragment flanked by the Barn HSV TK fragment act as "arms" as already? mentioned, having the DNA homologous to the DNA present in the L or S variants in which the modified F fragment is introduced. Again, due to an intracellular process in vivo, the mechanism of which is not? still known in its details, the F fragment of the modified HSV TK is incorporated into the vaccine variants within the cell, being then capable of replication and expression controlled by the vaccine virus.

This in vivo recombination technique finds wide application to the introduction of other foreign DNAs into the vaccine virus, thus offering a path for which the vaccine genome can? be modified to incorporate a wide range of foreign genetic material, including foreign DNA derived from vaccines, synthetic DNA or derived from organisms other than the vaccine virus.

Very different cells can serve as hosts for the aforementioned in vivo recombination. However, the efficiency of the recombination will vary? depending on the host cell. Among those investigated so far, the liver cells of the newborn Syriac hamster

[BHK-21 (Clone 13) (ATCC No.CCLIO)] showed the highest efficiency in the recombination processes. However, other cells including CV? 1 (ATCC NO. 'CCL70), a green monkey liver cell line, human TK cells (line 143) and a 5'-BUdR resistant mutant derived from human R970- 5, were also infected in this manner to generate vaccine mutants.

These cells should be suitably treated with the vaccine virus and foreign DNA for vaccine incorporation while for greater convenience the cells are found in the form of a monomolecular layer. For the purpose of in vivo recombination, the cells can be subjected to vaccine infection, then passing to the treatment with the foreign DNA to be incorporated into them, or they can first be put in contact with the foreign DNA, to then proceed with the vaccine infection. A further alternative is to expose the cells simultaneously to vaccine and foreign DNA treatment.

Viruses are conveniently placed in contact with the monolayer of cells in a common liquid medium, such as phosphate buffered saline, Hepes buffered saline or Eagle's special solution (with or without added serum), etc f as long as? compatible with such cells and viruses.

Conveniently, foreign DNA is used in the treatment of cells in the form of precipitated calcium phosphate. Are these methods for introducing DNA into cells already? Notes from earlier descriptions by Graham et al., Virology 52: 456-467, 1973.? Modified methods have been published by Stow et al. ', In J. Gen. Virol.?33:447-458, 1976 and by Wigler et al., In Proc. Nati. Acad. Sci. USA 76.:1373-1376, 1979.? The treatments suggested in these publications proceed suitably at room temperature, however the temperature conditions can be varied within certain limits compatible with viability. cellular, and the same applies to the duration of cellular treatment with the virus and / or the? precipitate of foreign DNA, resulting in varying degrees of efficiency in in vivo recombination. Also, the rate or degree of recombination will be? influenced by the concentration of the infectious vaccine virus and the amount of foreign DNA precipitate that is used. The other factors such as the atmosphere etc. are all chosen with the purpose of preserving vitality? cell phone. Apart from that, as long as? the three necessary constituents are present (the cell, the virus and the DNA), the in vivo recombination will proceed? at least within certain limits. The choice of optimal conditions for the particular case? certainly within the capabilities? experts in the microbiological arts.

Once the recombination phase has been completed, it is necessary to proceed with the identification of the mutated vaccine viruses by recombination in vivo, separating them from the unmodified ones.

Vaccine viruses mutated by recombination? ne in vivo of the foreign DNA can be separated from the unmodified ones with the alysilium of at least two methods, independent of the nature of the foreign DNA or of the ability? of the mutant to express any gene present in the foreign DNA. So, in the first place, foreign DNA can be found. identify in the mutant genome by genome restriction analysis in order to detect the presence of an extra piece of DNA in the mutated organism. analysis by restriction with the use of suitable restriction enzymes.1 By identifying the number of fragments and their molecular weight, we arrive at the structure of the genome prior to restriction by deduction. However, it is a laborious, time-consuming method and uncertain, given the need to cultivate purified platelets, and the risk that despite the numerous analyzes required, the cultured and analyzed platelets may be devoid of any mutant. ' A further method for detecting foreign DNA in the vaccine virus consists of a modification of the technique expounded by Villarreal et al, in the journal Science 196; 183-185, 1977. Here, the infectious virus is transferred from viral platelets found on the monolayer of infected cells, to a nitrocellulose filter. Appropriately, a mirror image of the virus transferred on the nitrocellulose filter is replicated, placing another such filter in contact with that side of the first nitrocellulose filter on which the viruses have been transferred. of the viruses on the first filter are thus transferred to the second one, one or the other of the filters, usually the first, is now used for hybridization.

The other filter is preserved to recover the recombinant virus, once the relative locus of the mate replicated on the mirror image filter has been identified, using the hybridization technique.

For the purpose of hybridization, the viruses found on the nitrocellulose filter are denatured with sodium hydroxide by means of already existing techniques. known. denatured is now hybridized with a radiolabeled counterpart of the gene whose presence we are trying to identify. For example, to identify the possible presence of vaccine mutants having the Barn HSV TK fragment, the corresponding fragment of the Barn HSV TK labeled with P is used, by analogy to the gi? Method. discussed in connection with the detection of modified plasmids due to the presence of this fragment. The unhybridized DNA is washed off the nitrocellulose filter and the remaining hybridized DNA, which? radioactive, is localized by autoradiography, that is to say by placing the filter in contact with a radiographic film.

Once the mutated viruses have been identified, the corresponding viral platelets are identified on the second filter, the one that contains the mirror image of the viruses transferred to the first filter, for the purpose of cultivation and replication of mutated viruses. '

The two methods just described involve the genotypic analysis of the organism in question and, how already? said, they can be used regardless of the possible expression of the gene present in the foreign DNA incorporated in the vaccine virus. However, when the foreign DNA is expressed, one can? use phenotypic analysis to identify mutants.

For example, if the gene manifests itself through the production of a protein having a relative antibody, the mutants can be detected by methods based on the formation of antigen-antibody complexes (see Bieberfeld et al. 1, J.?Immunol. Methods 6x 249-259, 1975). That is, the viral platelets containing the assumed mutants are treated with the antibody active against the protein produced by the vaccine genotype of the mutant. Excess antibodies are washed off the platelets, after which the platelets are treated with 125I-radiolabelled protein A. Protein A? able to form bonds with the heavy chains of the antibodies, thus constituting cos? a specific radiolab ature of the antigen complex, residual type on the monomolecular layer of the cell. the presence of radiolabeled immune complexes. This method is used to identify mutation vaccine viruses, capable of producing antigenic proteins.

In the particular case of foreign DNA accompanied by an HSV TK gene, once it is ascertained that the mutating vaccine virus expresses its own HSV TK gene, there is a much simpler method? simple and elegant to identify the presence of the gene. In fact, the ease? to distinguish between vaccine mutants carrying the HSV TK gene and the unmodified vaccine virus free from the gene itself, constitutes a powerful means of distinguishing vaccine virus mutants containing other exogenous genes present either alone in the vaccine genome or in combination with the HSV TK gene. ' These methods will be described in greater detail in more detail. beyond.

Since? eukaryotic cells have their own TK gene, so? as the vaccine virus possesses its TK gene (used as previously mentioned for the incorporation of thymidine into DNA), it becomes necessary to find some way to distinguish the presence and expression of these genes from the presence and expression of the HSV TK gene in vaccine mutants that are the subject of this discussion. To this end, we use the fact that the HSV TK? capable of phosphorylating halogenated deoxycytidine, pi? specifically iododeoxicitidine (IDC), a nucleoside, but n? the vaccine gene TK n? the cellular TK gene possess this phosphorylating action.-When IDC is incorporated into cellular DNA, it becomes insoluble. Conversely, the unincorporated IDC is easily eluted from cell cultures with an aqueous medium, such as a physiological buffer solution. Taking advantage of these facts, we proceed as follows to detect HSV TK gene expression in vaccine mutants.

The mononuclear cell layers are infected with mutation viruses in conditions capable of stimulating the formation of platelets, activating cell growth and l? viral replication. Infected cells are then treated with commercially-sourced radiolabelled IDC (IDC *), easily labeled with I. Purch? the cells have been infected with the virus containing the HSV TK gene, and provided? the gene itself is expressed, the cell prov eder? to incorporate the IDC * into their DNA. By washing the monomolecular cell layer now with the saline buffer, the non-incorporated IDC * will be released. eliminated by elution. If the monomolecular cell layers are then transferred to a nitrocellulose filter and exposed on a roentgen film, the film will result? opacified at the IDC * present in platelets, demonstrating the HSV TK gene expressed by vaccine mutants.

Using the aforementioned genotypic and phenotypic analyzes, the applicants were able to identify two vaccine mutants named VP-1 and VP-2.?The mutant VP? 1 (ATCC No. VR 2032 dep. 18-11-81)? a recombinant vickin virus derived from the vaccine variant S modified by in vivo recombination

with the plasmid pDP 13? 2. The VP-2 mutant (ATCC No.

VR 2030 dep. 18-1181)? the S variant of the vaccine virus, modified by recombination with the plasmid pDP 137.

Figure 4A constitutes a Hind III restriction map of the vaccine genome, indicating the HSV TK gene insertion site. Figures 4B and 4C illustrate on an enlarged scale the Hind III F fragment contained, respectively, in the mutants VF-1 and VP-2, to indicate the orientation of the Barn HI HSV TK fragment. Attention is drawn to the fact that in vivo recombination of pDP 137 with variant S

(ie VP-2) works to suppress one of the fragments

Barn HI HSV TK present in tandem pair in the plasmid of origin.-How already? previously mentioned, the fact that the HSV TK gene is expressed can serve for the quick and easy identification and identification of mutants with or without the HSV TK gene, or of a foreign gene that is present alone or in association with the HSV gene. The proof with its theoretical basis? described below.

The applicants succeeded in isolating a vaccine mutant in biologically pure form, and more? precisely a variant S, free from any functional TK gene of natural origin, which? been called VTK-79 (ATC-C NO.?VR 2031 dep. 18-11-81). As a rule, the S and L variants discussed above carry a TK gene in the Hind III J fragment. Where, the mutant in question, devoid of activity? of the vaccine gene TK, is used in the production of other mutation organisms endowed with the HSV TK gene, incorporated in the vaccine mutant with the methods already? described, the HSV TK gene present in these mutants will be? the only functional TK gene present in the virus / -The presence or absence of this HSV TK gene can be easily identified by culturing the cells infected by the virus on one of the various nutrients of choice.

One of the nutrients of choice contains bromodeoxyuridine (BUdR), a nucleoside analogous to thymidine for? with high mutagenic and toxic power to the organism, such as cellular or viral, if present in the DNA incorporated in the same.? Such a nourishing medium? been described by Kit et al., Exp / Celi Res / 31: 297-312, 1963 / Other mediums of choice are hypoxanthin / aminopterin / thymidine (HAT), Littlefield's medium, Proc / Nati. Acad. Sci. USA

50: 568-573, 1963 and related variants such as the MTAGG described by Davis et al., J. Virol / 13: 140-145, 1974 or another variant of the MTAGG described by Campione-Piccardo et al, in J. Virol. 3l_: 281-287, 1979. These nutrients are all electively capable of distinguishing between organisms containing an expressed TK gene and those containing no expressed TK gene.

The elective action of the medium? related to the factors described below.

There are two metabolic pathways for thymidine phosphorylation. The main one? independent of thymidine kinase and while synthesizing phosphorylated thymidine by means of intermediate mechanisms, not? capable of phosphorylating the BUdR n? to phosphorylate thymidine directly. The second metabolic pathway is based on the action of thymidine kinase and leads to the phosphorylation of both thymidine and its analogue, BUdR. the BUdR? a toxic substance of high mutagenic power, the presence of TK, for example the HSV TK in word, within the organism porter? to the phosphorylation of BUdR and its incorporation into the DNA of the living organism will result? in the death of the latter. Conversely, whether the TK gene is missing or not? expressed and the subsequent primary metabolic pathway leads to the synthesis of phosphorylated thymidine, but not to the phosphorylation of BUdR, then the metabolizing organism will be able to? survive in the presence of the BUdR, since? the latter is not incorporated into its DNA. '

The various growth behavior shown here is summarized in figure 5 reproduced in the annex, which shows the relative behavior and what? organisms in which 'TK is to be expressed * (TK <+>) and those without or not expressing the TK gene (TK) on a normal culture medium, or on an elective one such as HAT which blocks the primary metabolic pathway in the absence of the TK, or on one containing the BUdR. The TK + and TK ~ organisms will both grow in a normal nutrient due to the primary metabolic pathway independent of TK. In an elective nutrient such as HAT which blocks the primary metabolic pathway indi? pending from TK, the TK body nevertheless followed? its own growth as the enzyme prow e? der? to the phosphorylation required so that? the thymidine is incorporated in the DNA / Vice versa, the organism TK will not be able? Conversely, being organisms grown on a nutrient medium that contains the BUdR, the TK + variants do not survive, since the TK phosphorylation of the BUdR, and this toxic substance is then incorporated into the DNA. the BUdR not? phosphorylated via the primary metabolic pathway, the TK? I can? increase as the BUdR is not incorporated into the DNA.

Therefore, what if a vaccine virus devoid of vaccine TK, such as VTK 79? is used to insert the HSV TK gene according to the techniques described in this invention, it is easy to determine the presence and expression, or absence of the gene

HSV TK, by simply culturing the recombinants in an elective medium such as HAT. Will the mutation virus survive as it? they use the HSV TK gene to synthesize DNA.

In fact, the applicants were able to prepare several mutants of the vaccine virus without vaccine TK, named VP-3 (ATCC No. VR 2036 dep.'18-11-81), which? a recombinant r of VTK 79 and pDP 132, e

VP-4 (ATCC No. VR 2033 dep. 18-11-81), what? a recombinant of VTK ~ 79 and of pDO 137. The latter expresses the HSV gene that can be easily identified with the help of the already elective mediums? mentioned. 1

Two other recombination viruses, named VP? 5 (ATCC No. VR 2028 dep.-18-11-81) and VP-6

(ATCC No.?VR 2029 dep.?18-11-81) are respectively the recombinants of pDP 132 and pDP 137 with VTK ** 11 (ATCC No. VR 2027 dep. 18-11-81), which? a known vaccine variant L unable to express the vaccine TK gene, so? allows the introduction of DNA exceeding the maximum length of the vaccine genome.

The techniques exposed in this invention lend themselves to the introduction of the HSV TK gene in different pieces of the vaccine genome in order to identify the non-essential features of the genome itself, in other words, where the HSV TIC gene can be used. insert in the vaccine genome, cos? how does that region of the genome which has undergone the introduction fit into the Hind III fragment F? evidently not essential. Any non-essential site within the genome? a likely candidate for exogenous gene insertion, so that the methods set forth in the present invention are useful in building the maps of such non-essential sites within the vaccine genome.

Furthermore, if the HSV TK gene is paired with another exogenous gene and the resulting combination DNA material is introduced into a vaccine virus free of the vaccine TK gene, for example VTK 79, the recombinants that are formed and contain the foreign gene they will express the HSV TK gene and can be easily separated from the TK- * variants with the sorting technique just described.

Another embodiment of this invention relates to the preparation of a Hind III F vaccine fragment having an exogenous gene, as well as an exogenous gene. the treatment of the cells with this fragment together with a vaccine mutant unable to express the vaccine gene TK, but carrying the HSV TK gene incorporated by recombination in vivo, according to the techniques exposed in the present invention. labeling recovery and techniques used for incorporation of the modified Hind III F TK fragment into the vaccine virus discussed, 'cros-over' and recombinations may occur to produce another mutant in which the HSV TK modified F fragment is replaced from an F fragment having another exogenous gene. The resulting vaccine mutant, in which the HSV TK F fragment has been replaced by an F fragment bearing the exogenous gene, will be? completely devoid of TK, while the predominant virus, the one of origin not mutated, will be? always the HSV TK +.? Similarly, a foreign gene can? be inserted in the HSV TK gene present in this vaccine mutant, upsetting the integrity? of the gene and turning the recombinant organism into TK?, as opposed to the unchanged parent who remains TK.? In both cases,? It is easy to distinguish between vaccine mutants with the foreign gene and those without any TK, by cultivating them on BUdR medium and / or special nutrients such as HAT.

Understanding the invention and its multiple advantages can? take advantage of specific examples, cited here for illustrative purposes. Unless otherwise indicated, the percentages are referred to the weight. '

Example I - Isolation of Hind III vaccine fragments on agarose gelatin

The restriction Hind III endonuclease was purchased from Boehringer Mannheim. The DNA fermentative preparation was performed in 0.6 ml of Hind III buffer solution having a minimolar content of 10 mM Tris-HCl (pH 7.6 ), 50 mM NaCl, 10 mM MgCl2? 14 mM dithiothreitol (DTT) and 10 mcg / ml of BSA bovine serum albumin, containing 10-20 yig of vaccine DNA and 20-40 units? of Hind III (one unit corresponds to the quantity of enzyme sufficient to split 1 yig of X-DNA completely within 30 minutes).

Vaccine DNA was extracted and purified from viral particles (viri? Ni) as follows. The purified virions were disintegrated at a concentration equal to a density? 50 per ml optics, determined, at 260 nanometers (A260) 10 m d Tris? HC1 (PH 7,8), 50 mM ?? mercaptoethanol, 100 mM NaCl, 10 mM Na ^ EDTA, 1% Sarkosyl NL-97 and 26 % saccharose. Proteinase K was added at 100 µg / ml and the lysis product was incubated overnight at 37 ° C. The DNA was extracted by adding the same volume of phenol-chloroform in the ratio of 1: 1. The organic phase was removed by re-extracting the aqueous phase until a clear separation surface was obtained. Two more chloroform extractions followed after which the aqueous phase was thoroughly dialyzed against 10 mM of Tris-HCl (pH 7 , 4) containing 0.1 mM of Na3 EDTA at 4 ° C. The DNA was concentrated at about 100 µg / ml with Ficoll, a high molecular weight synthetic copolymer of sucrose and epichlorohydrin.

The fermentation of the DNA proceeded for 4 h at 37 ° C, then, to stop the reaction, it was heated to 65 ° C for 10 minutes, then adding an aqueous solution suitable to stop the reaction, consisting of agarose 2.5%, glycerin 40 %, sodium dodecyl sulfate 5% (SDS) and bromophenol violet 0.25% (BPB). The samples were applied in layers on agarose gelatin at 65 ° C and allowed to harden before proceeding to electrophoresis.

Electrophoresis was performed on 0.8% agarose gelatin (0.3 x 14.5 x 30 cm) in electrophoresis buffer containing 36 mM Tris-HCl (pH 7.8),

30 mM NaH2PO4 and 1 mM EDTA. Electrophoresis was done at 4 ° C for 42 h at 50 V. The gelatins were stained with ethide bromide (1 µg / ml in electrophoresis buffer solution). The restriction fragments were visualized with ultraviolet (UV) light to identify fragments to be cut from the gelatin.

To separate the fragments from the agarose gelatin we used? of the proceedings of Vogelstein et al., Proc. Nati. Acad. Sci. USA 7_6: 615-519, 1979 using vitreous dust as follows. The agarose gelatin containing the DNA fragment was dissolved in 2.0 ml of a saturated aqueous solution of Nal. 10 mg of vitreous powder was added for each] * g of DNA present according to the calculations. The solution was stirred overnight at 25 ° C to bind the DNA to the vitreous powder and thus the DNA. bound to the powder was collected by centrifugation at 2000 rpm for 5 minutes. The DNA-glass mixture was then washed with 5 ml of 70% Nal and collected again by centrifugation, by washing with a mixture of 50% buffer solution. [20 mM Tris-HCl (pH 7.2), 200 mM NaCl, 2 mM EDTA] and 50% ethanol. The DNA-glass mixture was again collected by centrifugation and carefully suspended in 0.5ml of 20mM Tris HCl (pH 7.2), 200mM NaCl and 2mM EDTA.

The DNA was then eluted from the vitreous dust at 37 ° C by incubation for 30 minutes. The glass was extracted by centrifuging at 10,000 rpm for 15 minutes. The DNA was recovered from the residue by ethanol precipitation, and was dissolved in 10 mM Tris-HCl (pH 7.2) containing 1 mM EDTA.

Fragment F isolated in this manner was used in the examples set forth below.

Example II - Insertion of the vaccine fragment

Hind III F in the Hind III site of the pBR 322 (construction of the pDP 3 fplasmid recombinant pBR 322 of the Hind III F vaccine fragment])

The Hind III F vaccine fragment was isolated from the preparation agarose gelatin as set out in Example I. This fragment was then inserted at the Hind III site of pBR 322 (Bolivar et al. Gene, 2: 95-113, 1977) as follows.

About 200 nanograms (ng) of pBR 322 were cleaved with Hind III in 10 mM Tris-HCl (pH 7.6). 50 mM NaCl, 10 mM MgCl2 and 14 mM DTT (Hind III buffer solution) using 1 unit? of enzyme for 1 h at 37 ° C. The reaction was stopped by heating at 65 ° C for 10 minutes. -500 ng of the isolated Hind III F vaccine fragment was added and the DNA was co-precipitated with 2 volumes of ethanol at -70 ° C for 30 minutes. then washed with 70% aqueous ethanol, dried and re-suspended in the ligating buffer solution consisting of 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 10 mM DTT and 1 mM adenosine triphosphate (ATP). About 100 units of T DNA binding (New England Biolabs) were then added and the mixture was incubated at 10 ° C overnight. The DNA treated with the ligase was then used for the transformation of E. coli HB101 (Boyer et al.?, J. Mol.> Biol.'41,: 459-472, 1969)

Example III - Transformation of E. Coli and selection of recombinant plasmids

Competent cells were prepared and transformed with plasmids according to the procedure described by Dagert et al., Gene 6: 23-28, 1979. E / coli HB101 cells were made competent by inoculating 50 ml of the LB broth (bact? tripton 1%, bacto-yeast extract 0.5% and NaCl 0.5% supplemented with 0.2% glucose) with 0.3 ml gives a culture of cells incubated overnight and cultured at 37 ° C as needed to register a density? optics (extinction) of 0.2 to 650 nanometers (A650) on the spectrophotometer. Then, the cells were chilled on ice for 10 minutes, centrifuged to form pellets, suspended again in 20 mL of cooled decimolar (0.1 M) CaCl2 and incubated on ice for 20 minutes. The cells were then pelleted and resuspended in 0.5 ml of cooled 0.1 M CaClg and left for 24 h at 4 ° C. The cells were transformed by adding bound DNA (or, 2-0.5 mg in 0.01-0.02 ml of ligating buffer) to 0.1 ml of competent cells. The latter were then incubated on ice for 10 minutes and at 37 ° C for 5 minutes. 2.0 ml of LB broth was then added and the cells were incubated at 37 ° C for 1 h with stirring. Aliquots of 10 or 100 microliters (??) were then spread on LB agar plates containing ampicillin (Amp) in a concentration equal to 100 pg / ml.

The transformation bacteria were then sorted to identify the recombinant plasmids, transferring the ampieillin-resistant colonies (Amp) to LB agar medium containing tetracycline (Tet) in a concentration equal to 15 pg / ml. Those colonies which were found to be resistant to ampicillin (Amp <R>) but sensitive to tetracycline (Tet) (approximately 1%) were tested for intact vaccine fragments of Hind III F inserted in pBR 322 according to the procedure suggested by Holmes et al., Anal. Bioch. 114? 193-197, 1981.

? 2.0 ml cultures of E_. coli transformation were cultured overnight at 3730. The bacteria were centrifuged to form pellets, suspended again in 105 µl of an 8% sucrose solution, Triton

X-100 5%, 50 mM EDTA and 50 mM Tris-HCl (pH 8.0) followed by the addition of 7.5 µl of a specially prepared lysozyme solution (worthington Biochemicals) [10 mg / ml in 50 mM Tris-HCl (pH 8.0)]. The lysates were placed for 1 minute in a boiling water bath, and then centrifuged for 15 minutes at 10,000 rpm. The residue was extracted and the DNA plasmid precipitated with the identical volume of isopropanol. The plasmids were again suspended in 40 ?? of Hind III buffer solution and fermented with 1 unit? of Hind III for 2 h. The pr? -4ot-ti-enzymatic digestion were then analyzed on a 1% agarose gelatin use analysis to identify the appropriate Hind III F fragments.

such "recombination plasmids, having an intact Hind III F fragment, termed the pDP 3, was used for further modification (see Figure 3B)."

Example IV - Preparative isolation of pDP 3

Large-scale isolation and purification

plasmid DNA was implemented by adapting the procedure of Clewel et al., Proc. Nati.?Acad. Sci / USA 62: 1159-1166, 1969.? 500 ml of LB broth was inoculated with 1.0 ml of E. coli HB 101 bacteria cultured overnight and containing the pDP 3. Density reached? optics of about 0.6 to A6? , 100 pg / ml of chloramphenicol was added to increase plasmid production (Clewel, J. Bacteriol. 110: 667-676, 1972). Bacteria were incubated at 37 ° C for another 12-16 hours, then collected by centrifugation at 5,000 rpm for 5 minutes, washed once in 100 ml of TEN [0.1 mM Tris-HCl (pH 8.0) buffer solution. , 150 mM NaCl and 10 mM EDTA], collected by centrifugation and suspended again in 14 ml gives a 25% sucrose solution in 0.05 M Tris-HCl

(pH 8.0). 4.0 mL lysozyme solution [5 mg / mL in 0.25 M Tris-HCl (pH 8.0)] was added and the mixture was incubated at room temperature for 30 minutes, before adding 4.0 ml of 0.25 M EDTA (pH 8.0) to it, the mixture was then placed on ice for 10 minutes. 2.0 ml of pancreatic RNase A (sigma Chemical Co.) [1 mg / ml in 0.25 M Tris-HCl (pH 8.0)] was added to this mixture which was then incubated at room temperature for 1 minute. The cells were then subjected to lysis by adding 26 ml of a lytic solution to the Triton [Triton x-100 1%, 0.05 M EDTA, 0.05 M Tris-HCl (pH 8.0)]. The mixture was incubated at room temperature for 30-60 minutes and the lysate was elarified by centrifugation at 17,000 rpm for 30 minutes at 4 ° C. the residue was then extracted and the DNA plasmid was separated from the chromosomal DNA on gradations of floating CsCl dye.

For this purpose, ethide CsCl bromide grades were prepared by dissolving 22 g of CsCl in 23.7 ml of clarified lysate. 1.125 ml of aqueous ethide bromide (10 mg / ml) was added to the solution. The mixture was then centrifuged in polyalomeric tubes in a Beckman 60 Ti mixer at 44,000 rpm for 48-72 h. The resulting DNA strands in the gradations were visualized under ultraviolet light and the lower strand, corresponding to the covalently closed DNA plasmid, was removed by piercing the tube with an 18 gauge needle connected to a syringe. The ethide bromide was extracted from the plasmid by repeated extraction with the double volume of isoamyl alcohol chloroform in the ratio of 24: 1. A thorough dialysis of the plasmids was then carried out against 10 mM of Tris-HCl (pH 7.4) containing 0.1 mM EDTA to eliminate the CsCl. The DNA plasmid was then concentrated by ethanol precipitation.

Example V - Construction of recombinant plasmids of pBR 322 / TK herpes vaccine virus

Figures 3B and 3C summarize the steps involved in the construction of the recombinant plasmids used for the insertion of the Barn HSV TK fragment in the S or L vaccine variants.

About 15 µg of covalently closed pDP 3 were cleaved by partial fermentation with Barn HI (Bethesda Research Laboratories) ie. Incubation in a Barn HI buffer solution, consisting of 20 mM Tris-HCl (pH 8.0), 7 mM MgCl2, 100 mM NaCl and 2 mM? -Mercaptoethanol, using 7 units? of Barn HI for 10 minutes at 37 ° C. Since? both the pBR 322 and the Hind III F vaccine fragment contain a Barn HI site, partial cleavage results in a mixture of interrupted linear plasmids at the pBR 322 site or that of the vaccine Barn HI. These mixed linear plasmids were then separated from the pDF 3 fragments cut at the two sites pBR 322 and the vaccine Barn HI by means of agarose gelatin electrophoresis and the linear plasmids? single cut were isolated by the vitreous powder method described in Example I.

A recombinant pBR 322 endowed with the 2.3 megadalton (md) HSV Barn HI fragment encoded for HSV TK, as exposed by Colbere? Garapin et al., Proc. Nati. Acad. Sci. USA 76_: 3755-3759, 1979, was brought to complete digestion with Barn HI and the 2.3 megadalton Barn TK fragment was isolated from the agarose medium as described above.

The recombinant pDP 3 Barn TK plasmids were constructed by binding approximately 1 µg of linearized pDP 3 per Barn HI to approximately 0.2 µg of the Barn TK fragment isolated in 20 µl binding buffer, containing 100 units. of DNA binding, at 10 ° C overnight. This binding mixture was then used to transform the competent cells of E. coli HB 101, as set forth in Example III.

Example VI - Sorting of transformation cells to identify those bearing recombinant plasmids equipped with HSV TK inserts

Transformation cells equipped with recombinant plasmids were subjected to sorting to identify HSV TK-inserts by hybridization of colonies, as essentially exposed by Hahahan et al., Gene 10: 63-67, 1980.

An initial set of nitrocellulose filters (Schleicher & Schull BA85) were placed on Petri dishes filled with nutrient LB agar containing 100 µg / ml of ampicillin. Transformation cells were applied to the filters and the capsules were incubated at 30 ° C overnight, or long enough to make the colonies barely visible. Did it happen? a replica nitrocellulose filter for each of the original filters, by superimposing a sterile nitrocellulose filter on each of the aforementioned original filters and pressing one firmly against the other. Then you practiced? on each pair of filters an incision with a sterile scalpel blade, after which the filters were separated and transferred to another LB agar plate containing ampicillin 100 pg / ml for 4-6 h. The original kit of filters was then placed on LB agar plates containing 200 µg / ml of chloramphenicol to increase plasmid production. Replication filters were stored at 4 ° C.

After 24 h on chloramphenicol, the original nitrocellulose filters were prepared for hybridization as follows. Each nitrocellulose filter was placed on a sheet of 0.5 N NaOH saturated vhatman filter paper for 5 minutes, dried with dry filter paper for 3 minutes and returned to the NaOH saturated filter paper for 5 minutes to induce bacterial lysis. and denaturing its DNA. This sequence was then repeated using vhatman filter paper saturated with 1.0 M Tris-HCl (pH 8.0) and repeated for the third time on filter paper saturated with 1.0 M Tris-HCl (pH 8, 0) containing 1.5 M NaCl for neutralization purposes. The nitrocellulose filters thus treated were then air dried and vacuum fired for 2 h at a temperature of 80 ° C.

Before proceeding with the hybridization, the nitrocellulose filters were then treated for 6-18 h by incubating them at 60 ° C in a pre-hybridization buffer solution. an aqueous mixture of 6 x SSC [1 x SSC = 0.15 M NaCl with 0.015 M sodium citrate (pH 7.2)], 1 x Denharts (1 x Denharts = a solution containing 0.2% Ficoll, BSA and polyvinylpyrrolidone, respectively) and 100-200 μg / ml DNA from salmon denatured cut sperm (S.S.DNA), 1 mM EDTA and 0.1% SDS. The treatment ? intended to reduce the quantity? of the binder between the filter and the non-hybridized DNA sample intended for application on the filter.

In the search for recombinant plasmids equipped with HSV TK inserts, the transformation colonies adhering to the original nitrocellulose filters so? treated, were hybridized with the P-labeled HSV TK Barn fragment by dipping the filters in a hybridization buffer containing 2 x SSC

(pH 7.2), 1 x Denhart solution, 50 pg / ml of S.S.

DNA, 1 mM EDTA, 0.1% SDS, 10% dextran sulfate 32

as well? P Barn TK as a hybridization probe. The level of radioactivity? of the solution was equal to a count of about 100,000 per minute (cpm) per milliliter.

Hybridization was completed at 60 ° C for 18-24 h (wahl et al., Proc. Nati. Acad. Sci. USA 76: 3683-3687, 1979).

[To prepare the hybridization probe, the 2.3 md Barn TK fragment was marked by translation of the incised portion according to the method of Rigby et al., J.

Mol, Biol. 113: 237-251, 1977. Precisely, it was ready? 0.1 ml of a reagent mixture containing 50 mM Tris-HCl (pH 7.6), 5 mM MgCl ^, 20 pM deoxicity triphosphate (dCTP), 20 pM deoxy triphosphate? adenosine (dATP), 20 pM deoxyguanosine triphosphate (dGTP), 2 pM (? - P) deoxytimidine triphosphate (dTTP) (410 curie / mMol) (Amersham Corporation), 1 ng of DNase I, 100 units? of DNA polymerase I (Boehringer Mannheim) and 1 µg of the Barn TK fragment. The reagent mixture was incubated at 14 ° C for 2 h. To surrender?

the reaction, 50 µl of 0.5 M EDTA were added, heated? giving it at 65? C for 10 minutes. The unincorporated triphosphates were extracted by gelatin filtration of the reagent mixture on Sephadex G50.]

After hybridization, the excess of the probing sample was extracted from the nitrocellulose filters by 5 washes in 2 x SSC (pH 7.2) containing 0.1% of

SDS at room temperature, followed by 3 washes in

0.2 x SSC (pH 7.2) containing 0.1% SDS at 60 ° C, each wash being continued for 30 minutes. The washed filters were then air dried and used

by exposure of X-ray film (Kodak x-omat R) at -70 ° C for 6-18 h, using a Soc. du Pont Cronex Lightening Plus screen to intensify the image.

After exposure and development, the radiographic film serves? then to identify the colonies containing the recombinant pBR 322 plasmids of the vaccine HSV TK Barn. The radiographic films were placed on the corresponding replica nitrocellulose filters. Colonies that tested positive were then collected by replication filters for further analysis.

Of the approximately one thousand colonies subjected to this sorting, 65 were found to be tentatively identified to carry the Barn TK insert within the pDP 3 plasmid.

Example VII - Restriction analysis of recombinant plasmids endowed with Barn HSV TK

Each of the 65 colonies tentatively identified as carriers of recombinant plasmids with the Barn HSV TK insert served? to inoculate cultures of 2.0 ml of LB broth containing ampicillin at the rate of 100 pg / ml. The cultures were then incubated at 37 ° C for one night, before plasmids were extracted as already. shown in example III. The plasmids were dissolved in 50? L d? Water after precipitation to isopropa? freight.

To ascertain whether the plasmids contained an intact 2.3 md Barn HSV TK fragment and at which Barn HI site within the pDF 3 Barn HSV TK had been inserted, 25 µl of each plasmid preparation was mixed with 25 µl of buffer solution. 2 x Hind III, and fermented at 37? C for 2 h with 1 unit? by Hind III. The resulting fragments were then analyzed by electrophoresis on a 1% agarose gelatin, as described above.

Of the 65 plasmid preparations so? analyzed, 6 resulted with the Barn HSV TK fragments inserted at the Barn HI site in the vaccine Hind III F tract of the plasmid, i.e., Hind III restriction fragments of a molecular weight equal to linear pBR 322 (2.8 md) as well as? fragments of a molecular weight higher than that of vaccine Hind III F fragments (8.6 md).

These 6 plasmids were subjected to further analysis with Sst I (an isoschizomer of Sac 1) to ascertain the number and orientation of the fragments

Barn HSV TK inserted in the Hind III F vaccine fragment, since? Sst I (Sac I) cleaves both the Barn HSV TK fragment and the Hind III F vaccine fragment asymmetrically. The analyzes were performed by mixing 25 µl of the plasmid with 25 µl of the 2 x Sst [50 mM Tris? HC1 buffer solution (pH 8 , 0), 10 mM of MgCl2, 100 mM of NaCl and 10 mM of DTT) and fermenting them with 1 unit? of Sst I (Bethesda Research Laboratories) at 37 ° C for 2 h. The resulting fragments were analyzed by electrophoresis on 1% agarose jellies. Of the six plasmids cos? analyzed, 5 resulted equipped with a pair of Sst I fragments having a molecular weight of 10.1 md and 3.5 md, indicative of a single Barn HSV TK insert.

One of these plasmids was selected for further investigation under the designation pDP 132. The other plasmid fru? three Sst I fragments with molecular weights of 10.8 md, 2.8 md and 2.3 md, indicative of tandem pairs of HSV TK Barn inserts oriented head-to-tail and of opposite orientation to that of pDP 132. was designated pDP 137. 'Figure 30? a diagram of the plasmids pDP 132 and pDP 137. '

Example VIII - Isolation of a TK *? S variant of the vaccine virus

To isolate a TK ~ S variant of the mutant vaccine virus, a viral population was subjected to high selective pressure to obtain such a mutant by culturing the virus in cells in the presence of the BUdR which? lethal to organisms carrying the TK gene. Pi? specifically, confluent monolayers of TK cells? humans (line 143) grown on Eagle's special nutrient in 150 mm Petn capsules were infected with about 3 x 10 <3> of unit? forming platelets (pfu, for 'plaque forming units') of variant S of the vaccine virus for each capsule (a total of 20), in the presence of 20? g of BUdR / ml. (Eagle's special nutrient is a commercially available medium for growing almost all cell lines. Alternatively other nutrients such as Eagle * s Minimum Essential Medium, Basai Eagle * s Medium, Ham * s-F1Q, Medium can be used. 199, RPMI-1640 etc.) Cultivation is carried out at 37 ° C in a CO2 enriched atmosphere. For greater convenience, you can? serve as a C02 incubator that delivers air sufficiently enriched with CO2 to have a concentration of approximately 5%.

Ninety-three of the developed platelets were isolated and re-plateleted on human TK cells (line 143) under the same conditions already. mentioned, again in the presence of 20? g of BUdR / ml / A certain number (5) of large and well isolated platelets were selected for further analysis /

The five isolated platelets were assayed for growth on monomolecular cell layers under the same conditions as previously used, with or without 20 µg BUdR / ml. The relative growth of each platelet with or without the BUdR was noted which was compared to the relative growth in similar cell cultures on monolayers by the S variant of the parent virus. The following results were obtained:

The growth of the material isolated from plate No. 79 was further examined at the presence of 0,

20 and 40 µg BUdR / ml and compared to the growth of the S variant of the parent virus. The following results were obtained:

In addition, the aforementioned five platelet isolates e

the S variant of the parent virus were controlled for growth on human TK <-> cells (line 143) in the presence of MTAGG that? a special nutrient from Eagle, modified for the presence of:

(see Davis et al. ', op.:cit.) which has elective action for thymidine kinase and against organisms lacking the thymidine kinase gene. to the following results:

Of the three materials isolated from platelets that exhibited complete inhibition of growth in the presence of MTAGG, isolate No. 79 was chosen arbitrarily and extracts from cells infected with virus No.? 79 were prepared to compare with extracts from uninfected cells. as well? from those infected with the s variant of the parent virus, to test its capacity? to phosphorylate thymidine to tritium (H). The following table summarizes the results: a

According to the following factors, (1) resistance to

BUdR, (2) growth inhibition on a medium containing MTAGG, and (3) lack of phosphorylation of some magnitude. of thymidine in infected cell extracts, it is assumed that the isolated N / 79 platelet material lacks activity. of thymidine kinase.

The insulated material carries the designation VTK 79.

Example IX - Marking recovery of variant L

of vaccine DNA by the S variant

Four preparations of the DNA variant L were prepared for the investigation of the labeling recovery.

The first consisted of the purified L variant and

intact vaccine DNA. The second preparation was

an L variant of vaccine DNA fermented with Bst

E II, a restriction endonuclease generating a

donor DNA fragment, fragment C, complete with

that DNA that is missing in the S variant but? just the variant L, being for more? accompanied at two o'clock

extremity of the DNA chain gives a region of the homol DNA

to the corresponding sequence in variant S. Preparation N / 3 and No. 4, respectively, consisted of DNA variant L fermented with Ava I and Hind III, which are restriction endonucleases capable of cleaving the vaccine genome within the sequence DNA characteristic of the L variant. The investigations on the recovery of labeling carried out with the four preparations in question show that the fragments of the L variant of the DNA accompanied by the deletion region that is missing in the S variant can be reintroduced in the S variant by means of a technique of in vivo recombination, provided? the fragment contains in addition to the deletion region also the terminal regions homologous to the corresponding sequence in the variant S. - For a better understanding of the fragments used in these investigations, reference is made to figures 6 A? C, since they are suddenly restriction maps of the left terminal of the vaccine genome. In particular, each map refers to the left end region of the genome comprising about 60 paired kilobases, as shown in the figure. That section of the vaccine genome which is suppressed in the S variant is represented in each map as the region between the dashed lines in the figures, equal in length to about 10 coupled kilobases.

With particular reference to figure 6AP? manifest that the fragment H obtained from fermentation with Ava I? completely located within the region of deletion, for? lacks terminal DNA fragments homologous to variant S DNA due to the fact that the Ava I cleavage sites are located entirely within the suppressed region in variant S.

The restriction map shown in Figure 6B relative to Hind III shows that this restriction enzyme also fails to produce the DNA fragment of the L variant homologous to the suppressed region in the S variant. In this case, the sequences homologous to the S variant are found at the left terminal of the C fragment of the Hind III. However, the restriction site at the right terminal of the C fragment? located within the deletion region, lacking instead a terminal sequence homologous to the DNA sequence of the S variant. fragment C, complete with the region of deletion which is missing in the variant S, and also equipped with terminal sections at the end? right and left, which are homologous to the corresponding section of the variant S.?

As discussed below, the results of these investigations show that the DNA present in the L variant but suppressed in the S variant can? be recovered from variant S with a high degree of efficiency from the intact genome of variant L and with less efficiency from the C fragment of Bst E II; 'instead, can not? be recovered from the L variant DNA fragments prepared with the restriction endonucleases Ava I and Hind III.

The high degree of efficiency with which the suppressed sequence is recovered from the intact variant L can? attributing to the fact that only one interchange (crossover) between the intact L variant and the S variant is enough to produce a genome of the variant type L. On the other hand, to recover the trait suppressed by the .C fragment of the Bst E II, it is necessary an interchange between the fragment and the variant S in both the left and right terminal tract of the fragment C, in order to incorporate the deletion region in the variant S. Finally, since? n? fermentation with Ava I n? fermentation with Hind III pu? producing DNA fragments capable of incorporation into the S variant via any interchange, no recovery of the suppressed trait occurs.

Marking recovery was performed on CV-1 monolayers using the calcium phosphate technique expounded by Graham et al., Virology 52: 456-467, 1973 in the modification by Stow et al, and Wigler et al., Already? mentioned above. For this purpose, confluent monolayers of CV-1 were infected with variant vaccine virus S to create approximately 50 - 200 platelets in a number (5-20) of 6 cm diameter Petri dishes. To infect the cells, the nutrient (e.g. Eagle special medium containing 10% calf serum) is aspirated and a virus dilution containing 50-200 pfu / 0.2 ml in a cell compatible nutrient, e.g. Eagle's Special with 2% calf serum, is applied to the monolayer of the cell.

After a one-hour incubation period at 37 ° C in a CO2 incubator to allow the cells to absorb the virus, several preparations of the L variant of DNA, among the four mentioned above, were separately added to the monolayers. in the form of a precipitate of calcium phosphate containing for each capsule a microgram of the preparation of the L variant of the DNA. After 40 minutes Eagle's special nutrient with 10% calf serum was added and four hours after the initial DNA addition, the monolayer cell layer was exposed to 1 ml of 25% buffered dimethyl sulfoxide for four minutes. The buffer solution contains 8 g of NaCl, 0.37 g of KC1, 0.125 g of Na2HP0 .2H20, 1 g of dextrose and 5 g of N- (2-hydroxyethyl) piperazine, N '- (2-ethanosulfonic acid) ( Hepes) per liter, with a final pH level of 7.05. The dimethyl sulfoxide was extracted and the monolayers were washed and topped with an agar nutrient layer. After three days in a CO2 incubator at 37 ° C, the cells were stained with an agar nutrient layer containing a neutral red dye suitable for staining uninfected cells (the agar nutrient = Eagle's special nutrient containing 10% yolk serum and 1% agar). The next day the agar layer was removed and the monomolecular layers were transferred to nitrocellulose filters, where they were prepared by in situ hybridization according to the method of Villarreal et al., Loc. cit. Since? fermentation of the L variant genome with Ava I generates a paired fragment of 6.8 kilobases, the H fragment residing entirely within the singular suppressed DNA sequence in the S variant genome (see Figure 6A), the Ava I H fragment labeled at <32> P and translated by incision provides a highly specific probe to detect recovery of the single DNA sequence of the -variant L by that of S.

For hybridization the nitrocellulose filters were placed between cut-out circles of Whatman No.1 filter paper in Petri dishes with a diameter of 6 cm and pre-hybridized for 6 h at 60 ° C in a pre-hybridization buffer solution (SSC, Denhardt's solution, EDTA and S.S.DNA), as already? previously described in Example VI. The radioactive probe constituted by the Ava I H fragment of the variant L, marked at <32> Pe translated by incision, having an activity? specification of approximately 1 x 10 <8> cpm / pg was used for hybridization in 2 x SSC, 1 x Denhardt, 1 mM EDTA, 0.1% 3DS, 10% dextran sulfate and 50 yig / ml of S.S.DNA treated with sound waves, overnight at approximately 1 x 10 cpm / ml at 60 ° C. The radioactive probe was prepared according to the method of Rigby et al., J. Mol. Biol. 113: 237-251.1977. The filters were repeatedly washed at room temperature and 60 ° C using the washing procedure described in Example VI, then air dried and radio-autographed.

The results of these tests are summarized in the following Table I.

No less than 5,000 platelets were analyzed for each DNA donor preparation.

Example X - In vivo recombination using pDP 132 and pDP 137 to generate mutants of the vaccine virus VP? 1 up to VP-6 and the relative identification

with the help of replication filters

A first precipitate of calcium orthophosphate of

Donor DNA was prepared by combining 5µg of DNA fermented with pDP 132 Hind III in 50µl of water, 4µg

of the S variant carrier DNA (prepared according to Example I) in 40 µl of water and 10 µl of 2.5 M CaCl2, combining the resulting mixture with an equal volume of

2 x Hepes phosphate buffer comprising 280 mM NaCl, 50 mM Hepes and 1.5 mM sodium phosphate (pH 7.1) and leaving the precipitate to form for a period of 30 minutes at room temperature. A secon

do precipitate was prepared in the same way? and with identical quantities, except that the DNA was that fermented with pDP 137 Hind'III,

(According to the more detailed description by Stow and coll. Loc.cit. And Wigler and coll., Loc.cit., The modifications of the Graham et al. Precipitation technique, cited above, use carrier DNA as a high weight compound molecular to increase the efficiency of the precipitate formation of calcium orthophosphate. The carrier DNA that is used is that coming from the virus used to infect the cells of the monolayer according to the in vivo recombination technique.)

For in vivo recombination purposes, confluent monolayers of CV-1 cultured on Eagle's special nutrient containing 10% calf serum were infected with the S variant of the vaccine virus at an infectious titer of a multiple of 1 pfu / cell. The infectious process? analogous to that of Example IX. The virus was allowed to absorb for 60 minutes at 37 ° C in a CO2 incubator, after which it was aspirated. the inoculation material and lav? the monomolecular cell layer. The precipitated DNA preparations were applied on separate cell layers and after 40 minutes in a C02 incubator at 37? Eagle's special medium containing calf serum? up to 10%. In each case, the virus was collected after 24 hours at 37 ° C in the CO2 incubator by three freeze / thaw cycles, and minced on monomolecular CV-1 layers. Approximately 15,000 platelets on CV-1 monolayers were tested for recombination virus using replication filters prepared as described below.

Platelets formed on confluent monolayers of CV-1 beneath the agar nutrient layer were transferred to nitrocellulose filters, gently removing the superimposed agar layer with a scalpel and placing the nitrocellulose filters on the monolayer. To establish adequate contact between the filter and the monolayer, sheets of Whatman No. 3 filter paper, moistened with 50 mM of Tris buffer (pH 7.4) and 0.015 mM NaCl, were placed on top of the nitrocellulose filter and pounded with a cap. of rubber until the monomolecular layer transferred onto the nitrocellulose showed a uniform color around distinct non-colored areas on the platelets. (The monomolecular layer was previously stained with neutral red which is absorbed by viable cells, ie those that have not undergone lysis due to viral infection).

The nitrocellulose filter bearing the transfer monolayer is now removed from the Petri dish and placed with the monolayer side up. Another nitrocellulose filter moistened in the aforementioned Tris-NaCl solution is now placed directly on the first filter and the two are firmly placed in contact, stamping them with a rubber stopper, having previously protected the filters with a circle cut out of Whatman No. 3 paper? . Once the filter paper is removed, the nitrocellulose filters are notched by orientation and then separated. At this point the second nitrocellulose filter (the replica) contains a mirror image of the monomolecular cell layer transferred to the first nitrocellulose filter. For convenience, the second filter is placed in a clean Petri dish and frozen. The first filter is hybridized using the <32> .P-labeled HSV TK Barn fragment as a probe. The preparation of the probe and the hybridization technique were described in Example VI.

Approximately 0.5% of the platelets tested for hybridization were positive, ie they were the recombinant virus with the Barn HSV TK gene.

The recombinant virus platelets corresponding to those identified in the first nitrocellulose filters by means of hybridization were then isolated.

from replication nitrocellulose filters with the following technique, for the purpose of further purification.

Using a cork drill having a slightly larger diameter of the plate to be picked up, the selected plate is punched from

first nitrocellulose filter, the original one used for the identification of recombinant viruses by hybridization. The resulting perforated filter is then used as a matte to locate and isolate the corresponding chip on the replication filter. Precisely, the replication filter is placed with the

monomolecular side facing up, over a sterile surface, and covered with a Saran plastic sheet.

The first or original perforated filter is now placed with the respective monolayer facing

above the second filter, and the orientation notches marked on the filters are aligned to match the mirror images on the plates.

Always using the cork drill, a cap is removed from the replica filter, the protective sheet of Saran is removed and placed in 1 ml of special nutrient of

Eagle containing 2% calf serum. The nitrocellulose cap is treated in this medium with sound waves for 30 seconds on ice to release the virus.

0.2 ml of this viral preparation and 0.2 ml of a 1:10 dilution of the preparation itself are applied on the monolayers CV-1 in the 6 cm diameter Petri dishes.

By way of purification of the platelet, the entire sequence of preparation of the first nitrocellulose filter, replication filter, hybridization and isolation of the platelet from the replication filter was then repeated.

A sample of the purified platelet prepared in this manner from a calcium d orthophosphate precipitate of DNA fermented with Hind III pDP 132 was termed the vaccine virus VP-1. By analogy, a platelet containing the recombinant virus prepared from DNA fermented with PDF-137 Hind III was named VP2. Both samples were cultured on suitable cell media for further investigation, including identification by restriction analysis and other techniques. ?

Similarly, two other vaccine mutants were prepared, respectively named VP-3 and VP-4, by in vivo recombination using VTK ~ 79 (an S variant of the TK vaccine virus already described in Example VIII) as recovery virus and of pDP 132 and PDP 137, respectively, which pia donor DNA? smidi. The precipitates were formed as already? described above, except that 5 µg of plasmid donor DNA contained in 50 µl of water, 4 µg of VTK carrier DNA ~ 79 in 150 µl of water and 50 µl of 2.5 M CaC12 were combined and added drop by drop to an? equal volume of 250 ?? of the Hepes phosphate buffer described above.

Furthermore, the cells chosen for infection by the viral carrier VTK-79 were those BHK-21 (Clone 13) instead of CV-1.

Two other vaccine virus mutants named VP-5 and VP-6 were prepared using calcium orthophosphatic precipitates of pDP 132 and pDP 137, respectively, prepared similarly to mutants VP-3 and VP-4. However, in the case of the VP-5 and VP-6 mutants, the carrier DNA was the VTK ~ 11 vaccine virus instead of the VP-5 and VP-6 mutants. VTK 79.

Here too, the infected monolayers were those of BHK-21 (C-13) cells, while the recovery virus was VTK ~ 11 in this case.

Example XI - Expression of HSV TK by the vaccine mutant VP? 2 - and the use of IDC? * For its identification

The viral product obtained in Example X by the in vivo recombination of the variant s of the vaccine virus and the calcium orthophosphatic precipitate of the DNA fermented with pDP-137 Hind III was spread on confluent monomolecular layers of CV-1 cells present in about twenty Petri dishes of 6 cm, at a concentration sufficient to form approximately 150 platelets per capsule. The platelets were covered with a liquid nutrient, for example? Eagle's special medium containing 10% calf serum. After 24 to 48 hours of incubation at 37 ° C in a CO2 incubator, the liquid cover nutrient was removed from the capsules and replaced in each case by 1.5 ml of the same liquid cover medium containing 1? 10? Ci <125> I labeled iododeoxycytidine (IDC). The plates were then incubated overnight at 37 ° C in a C02-rich atmosphere, after which the monomolecular cell layer was stained with the addition of neutral red to visualize the platelets by contrast.

At this point the nutrient was removed by aspiration, the monomolecular layers were washed three times with a phosphate buffered saline solution, and the monomolecular layer of the cells on each plate was imprinted on a corresponding nitrocellulose filter. The latter? it was then exposed to roentgen film for 1 to 3 days, and then developed.

- --- Those viral platelets which contain and express the HSV TK gene are capable of phosphorylating the IDC and incorporating it into their DNA, making the latter insoluble. The non-phosphorylated and non-incorporated IDC * residue was washed off, so the platelets that opacify the radiographic film are those capable of expressing the recombinant HSV TK gene. N? the CV-i n cells? the vaccine virus, although endowed with TK, are capable of phosphorylating and incorporating IDC * in the elective mode characteristic of the HSV TK gene.

After identification of the recombinant organism by means of radioautography, filter caps were cut out of the nitrocellulose filters, which were then placed in 1 ml of the covering nutrient (Eagle's special medium containing 10% calf serum), treated with waves. sonorous and re-smeared on the monomolecular layers of CV? 1. The LSD * assay was then repeated to further purify the isolated viral material. In this way, a virus identical to the VP-2 mutant identified by hybridization in example X was isolated by a technique based on the expression of the HSV TK gene present in the same. ?

Again, the results of this example demonstrate the expression of the HSV TK gene present in recombinant organisms. to the present invention, by means of some of these organisms.

The vaccine mutants derived from pDP 137, namely VP-2, VP-4 and VP-6 are all capable of expressing the HSV TK gene present in them by phosphorylation and incorporation of lDC *, in the manner described above. Conversely, the variants VP? 1, VF-3 and VP-5 derived from pDP 132 are not capable of expressing the gene, possibly due to the fact that the orientation of the gene within the virus? opposite to the direction in which the gene is transcribed.

Example XII - The use of an elective medium for the identification and isolation of recombinant viruses containing the HSV TK gene

The viruses prepared according to Example X for in vivo recombination within the BHK-21 (C-13) cells of the vaccine virus VTK ~ 79 and an orthophosphatic precipitate of calcium of the pDP 137, were used to infect human TK cells (line 143). Pi? specifically, monomolecular cell layers placed in five Petri dishes of 6 cm in diameter, were infected with the virus described in Example X, in a viral dilution of 10 to 10 in the presence of the elective nutrient MTAGG.

Was the infectious technique identical to that already? exposed.

Five well separated platelets and one were isolated

Claims (1)

1. A vaccine virus modified by the presence of exogenous DNA in the vaccine genome. A vaccine virus according to claim 1, wherein said exogenous DNA is expressed in a host organism by means of the production of a protein. 3. A vaccine virus according to claim 2, wherein said protein? an antigen.? 4. A vaccine virus according to claim 2, wherein said exogenous DNA? a herpes simplex gene expressed in a host organism by means of thymidine kinase production. 5. A vaccine virus according to claim 4, wherein the aforementioned virus? devoid of the vaccine gene producing thymidine kinase. 6.1 The vaccine virus VP-1 according to claim 1. The VP-2 vaccine virus according to claim 1. The VP-3 vaccine virus according to claim 1. The VP-4 vaccine virus according to claim 1. 10. The VP-5 vaccine virus according to claim 1. * The VP-6 vaccine virus according to claim 1. 12. A method for in vivo recombination of vaccine virus and donor DNA which includes contacting a monomolecular cell layer with a culture medium compatible with the cell containing the aforementioned vaccine virus and with a compatible culture medium with the cell containing the aforementioned donor DNA, said donor DNA having the exogenous DNA present within an otherwise co-linear segment of the vaccine DNA in a non-essential region within that segment. 13. A method according to claim 12, in 21. A method according to claim 20, wherein said DNA is expressed by that cell. 22. A method according to claim 21, wherein said DNA is expressed by the production in this cell gives a biological substance. 23.-A method according to claim 22, in which the aforesaid DNA to be replicated contains the herpes simplex TK gene and the aforesaid biological substance? thymidine kinase. 24.1 A recombinant plasmid comprising pBR 322 DNA in one with a member selected from a group consisting of the Hind III F vaccine DNA fragment and at least one Bam HI HSN TK fragment. 25. A plasmid according to claim 24, which? the PDP 3. 26 / A plasmid according to claim 24, which? the PDP 132. 27. A plasmid according to claim 24, which? the PDP 137. The method of augmenting a protein comprising inoculating the host animal, susceptible to the vaccine virus, with a vaccine virus according to claim 2. 29. The method of augmenting a protein comprising inoculation of the host animal, susceptible to which the above "otherwise co-linear" segment of the vaccine DNA is clonable with a plasmid. A method according to claim 13, wherein the aforementioned co-linear segment of the vaccine DNA? the relative fragment Hind III F. ' A method according to claim 14 wherein said exogenous DNA includes the herpes simplex Barn HI TK gene. 16. A method according to claim 12, wherein said donor DNA? present in the form of calcium orthophosphatic precipitate of said donor DNA. 17. A method according to claim 12, wherein the aforementioned vaccine virus? devoid of a vaccine gene producing thymidine kinase. 18 / A biologically pure culture of the vaccine virus VTK ~ 79.? 19. The method of DNA replication in a eukaryotic cell by infecting said cell with a vaccine virus modified to contain such DNA, the DNA being exogenous to the aforementioned vaccine virus. 20. A method according to claim 19, wherein the aforementioned DNA? equally exogenous to said cell. to the vaccine virus, with a vaccine virus according to claim 3.? 30. ' The method of immunization of the host animal, susceptible to the vaccine virus, to develop antibodies against an antigen, which method comprises the inoculation of the host animal with a vaccine virus having within the vaccine genome the DNA exogenous to that genome and encoded for the said antigen.